The broad goal of this proposal is to identify specific amino acid residues critical for syntaxin 1A-CFTR interactions and to use this information to augment C1- current activity in vivo and ex vivo by maneuvers that disrupt their interactions with syntaxin 1A in whole animals (WT, G551D and R117H CFTR mice). Our preliminaiy results indicate that syntaxin 1A binds to G55 1 D CFTR and inhibits its activity in Xenopus oocytes. In addition, syntaxin 1A is present in native tissues affected in CF (in particular, airway epithelium), and we will show that an 18 a.a peptide of CFTR (p46-63) that can block the syntaxin 1A-CFTR interaction can augment CFTR activity in polarized colonic epithelial cells (HT29-CL1 9A). We have preliminary data to indicate the feasibility of these experiments in whole animals as well. A few organic molecules have been screened and we have identified one positive hit (SRI 1725) that can disrupt syntaxin 1A-CFTR interaction. Functional data suggesting an augmentation of CFTR activity upon pre-treating the cells by this molecule will be presented. These preliminary data form the basis for this proposal. The main aims of this project are (1). To identify residues that are essential for syntaxin 1A-CFTR interaction and to disrupt this interaction in order to augment the activity of WT and partial loss of function CFTR mutants in whole animals. (2). To identify small organic molecules that disrupt CFTR-syntaxin 1A interaction. (3). To determine the specificity of these organic molecules on other syntaxin 1A interacting proteins (SNAP-23, VAMP-2, Munc-18, Calcium channels and ENaC) and to monitor the effects of these organic molecules (that specifically disrupt only syntaxin 1A-CFTR interaction) in whole animals. These studies should help us identify critical residues involved in CFTR and syntaxin 1A interaction. Also, these studies may help us understand how syntaxin 1A down regulates the C1- channel function in whole animals. These experiments will also provide a test of the concept that mutant CFTR function can be potentiated by maneuvers that block this protein-protein interaction. Finally, these studies should help us identify a few novel cell permeant organic molecules that can augment CFTR activity of partial loss of function CFTR mutants that will eventually be useful in CF therapy.